Chirality is a key factor in the efficacy of many biomolecules which results in different stereoisomers having different biological activities. There is an increasing demand in the production of optically pure compounds due to regulatory requirements, prospects of lower toxicity and exquisite efficiency. Biocatalysis often referred to as a green chemistry approach, is widely researched and has developed into a standard technology for the production of enantiopure compounds. Enantiopure epoxides and diols are important chiral building blocks for pharmaceuticals and agrochemicals. A major challenge in the conventional organic synthesis is to generate optically active compounds with high enantiopurity and yield.
Several chemical procedures have been developed for the synthesis of optically active precursors like Sharpless-epoxidation with hydroperoxides and metal catalysts, which is limited to allylic alcohols [Katsuki, T. and Sharpless, K. B. (1980) J. Am. Chem. Soc. 102, 5975-5976]. Similarly, Jacobsen's asymmetric epoxidation using optically active (salen) manganese (III) complex is limited to steric and electronic nature of catalysts and prefers cis-alkenes [Jacobsen, E. N., Zhang, W., Muci, A. R., Ecker, J. R. and Deng, L. (1991) J Am. Chem. Soc. 113, 7063-7064]. The biological process includes direct stereoselective epoxidation of alkenes by monooxygenases [Archelas, A. and Furstoss, R. (1999) Topics Curr. Chem. 200, 159-191], and a two step synthesis using haloperoxidases and halohydrin epoxidase [Besse, P. and Veschambre, H. (1994) Tetrahedron 50, 8885-8927] or using other biocatalytic enzymes like and alcohol dehydrogenase and lipase [Cipiciani, A., Cittadini, M., and Fringuelli, F. (1998) Tetrahedron 54, 7883-7890]. However, lipase-mediated hydrolysis sometimes results in instability of the final product and further irreversibly denatures the enzyme. [Braun, B. and Klein, E. (1996) Biotechnol. Bioeng. 51: 327-341.] [Hasegawa, J., Ogura, M., Tsuda, S., Maemoto, S., Kutsuki, H. and Ohashi, T. (1990) Agric. Biol. Chem. 54, 1819-1827]. Many of these processes suffer from significant drawbacks like limited substrate scope, or efficient only at low concentrations, use of expensive and toxic metal catalysts, limited efficiency and productivity with varying degrees of enantioselectivities.
In the recent years, epoxide hydrolases have emerged as promising biocatalysts which offer a relatively simple route for the synthesis of optically enriched epoxides and diols under mild and eco-friendly conditions. This enzymatic resolution converts the inexpensive and easily available racemic mixture of aryl epoxides into optically active epoxides with excellent enantiomeric excesses. Aryl epoxides and the related compounds are potential intermediates for synthesis of chiral amino alcohols [Kamal, A., Chouhan, G. (2005) Tetrahedron Asymmetry 16, 2784-2789] and β-blockers [Kamal, A., Sandbhor, M., and Shaik, A. A. (2004) Bioorg. Med. Chem. Lett. 14, 4581-4583].
Epoxide hydrolase [EC 3.3.2.3] is a hydrolytic enzyme comprising of functionally related amino acid residues which catalyze the trans antiperiplanar addition of water to oxirane compound generating vicinal diol. The chiral substituted epoxides as well as the diols are valuable precursors in the downstream synthetic steps. Optically pure epoxides have gained attention as they are a common structural element in both simple and complex bioactive compounds and due to their electronic polarization and versatility of the oxirane function; they can be transformed into valuable synthons. Epoxide hydrolases are ubiquitous in nature and found in majority of organisms populating every branch of the evolutionary tree. They are found in majority of the mammals, plants and microbes. Mammalian epoxide hydrolases have been extensively studied. Inspite of its exceptionally high enantioselectivity for a large substrate spectrum, limited availability has hampered its large-scale production.
Microbial epoxide hydrolases have emerged as a new biotechnological tool for green synthesis of optically pure synthons. This environmentally compliant methodology is attractive as it minimizes the costs of resources and prevents the production of toxic waste in industrial applications. Over the past decade many epoxide hydrolases have been explored from microbial origin, but most of these enzymes have a limited substrate scope or rather act on low substrate concentrations due to low catalytic efficiency of the enzyme. Relatively better enantioselectivities were obtained from fungal epoxide hydrolases (Archelas et al. 1993; Nellaiah et al. 1996; Pedragosa 1997), but they have experimental constrains like inhibition at high substrate concentration, low enzymatic activity (Pedragosa et al. 1993). The mycelial and filamentous fungi are often characterized by high broth viscosity, nutrient concentrated zones, insufficient oxygen and mass transfer which reduces the productivity, high substrate concentrations cannot be usually used as they are inhibitory for the reaction and the reaction kinetics cannot be measured accurately as the insoluble substrates adsorb onto the mycelium, therefore the epoxide hydrolases require partially or purified enzymatic preparations for preparative scale experiments, however enzymatic preparations at higher substrate concentrations get deactivated or become less enantioselective at later stages (Morisseau et al. 1997 Liu et al. 2006).
Some of them have been cloned and functionally expressed to meet the growing demand via preparative scale application, for example, Rhodotorula mucilaginosa expressed in Yarrowia lipolytica [Labuschagne, M. and Albertyn, J. (2007) Yeast 24, 69-78]. Similarly, Aspergillus niger and Rhodococcus erythropolis expressed in Escherichia coli [Bottalla, A.-L., Ibrahim-Ouali, M., Santelli, M., Furstoss, R., and Archelas, A. (2007) Adv. Syn. Catal. 349, 1102-1110]. However, the substrate range of these enzymes in terms of their enantioselectivity has recently been examined. Higher enantioselectivity has been observed in limited number of organisms and only two organisms (Aspergillus niger and Rhodococcus rhodochrous) have so far been commercialized. Most of the known enzymes have limited substrate scope or rather act at low substrate concentrations due to low catalytic efficiency of the enzyme.
Majority of the epoxide hydrolases from various microbes reported to-date cannot tolerate high substrate concentrations and become less enantioselective during the biotransformation process. For example, in the case of Sphingomonas sp. HXN-200 the hydrolase can carry out the hydrolysis of styrene oxide at 320 mM, which yielded 40.2% with a final concentration of 128.6 mM of S-styrene oxide (S-enantiomer). And after 13.8 h the reaction velocity decreased and hydrolysis became less enantioselective. However, the epoxide hydrolase from Achromobacter sp. MTCC 5605 performs the hydrolysis of styrene oxide at 500 mM, which yielded 41.8% with a final concentration of 209 mM of S-styrene oxide. Further, the biotransformation process carried out by Sphingomonas sp uses a cell-free extract which required pre-processing involving the preparation of cell-free extract by passing the cells through French press, ultracentrifugation for cell debris removal followed by lyophilization of the cell-free extract which is cumbersome and cost-intensive, and the stability of the enzyme is less (only for 13.8 h). However, the present method using the epoxide hydrolases from Achromobacter sp. MTCC 5605 is cost-effective as there are no pre-processing steps involved, since it is a whole-cell biotransformation which allows continuous hydrolysis of epoxide substrates even at high concentrations and thereby the kinetic resolution process (biotransformation) can be prolonged for more than 48 h until the final chiral epoxide with high enantio-purity is obtained. Moreover, the lyophilized whole-cells can be stored for longer duration without any loss of activity and are abundantly available and accessible to the organic chemist.
The bacterial epoxide hydrolases reported in the prior art are referred to as below:                (1) Bacillus megaterium ECU1001 hydrolyses phenyl glycidylether which has an E value of 47.8 for a substrate concentration of 60 mM and yield of 25.6% [Tang, Y.-F., Xu, J.-H., Ye, Q. and Schulze, B. (2001) J. Mol. Catal. B: Enzymatic 13, 61-68].        (2) Sphingomonas HXN 200 hydrolyses styrene oxide at 320 mM concentration with an E value of 26-29 and yield of 40% [Liu, Z., Michel, J., Wang, Z., Wilholt, B. and Li, Z. (2006) Tetrahedron: Asymmetry 17, 47-52].        
The usefulness of epoxide hydrolases as biocatalysts that produce enantioenriched epoxides is dependent on the factors like activity, availability, stability of the enzyme. The active whole cells can be used in lyophilized form for resolution of pharmaceutically important epoxides with high enantioselectivity. The interesting properties of this epoxide hydrolase has been justified further by exploring the biotransformation conditions like growth and epoxide hydrolase production, the ratio of substrate to biocatalyst concentration and the other physico-chemical properties like effect of temperature, pH, solvents, metal salts have been studied to increase the efficiency of the enzyme. In view of the non toxicity, easy availability and low cost, the whole cell catalysts i.e. bacterial epoxide hydrolases provide green and economical synthesis of optically pure synthons which can provide valuable methods for preparative scale applications.
In view of the above facts, there is an urgent need to provide a highly enantioselective epoxide hydrolase enzyme with a high catalytic efficiency and expanded substrate spectrum. It may be noted that a highly enantioselective bacterial epoxide hydrolase that belongs to the family Alcaligenaceae and genus Achromobacter, has not been previously documented. In majority of the bacterial epoxide hydrolases, it has been reported that they have low substrate tolerance, and limited substrate spectrum with low reaction rates, and therefore need further cloning or be functionally expressed to meet the growing demand for chiral biotransformations. The present invention fulfils these requirements as it provides a novel bacterial strain of Achromobacter sp. producing a novel epoxide hydrolase which functions even at high temperatures of up to 50 degree C. and pH of 9.0, which is a first report of its kind. Further, it is pertinent to mention here that whole cell resolutions allow continuous hydrolysis for high substrate concentrations; can be easily cultured, abundantly available and accessible to organic chemists. Therefore, there is an urge to develop a novel epoxide hydrolase for an efficient catalysis with expanded substrate spectrum and a cost effective process for practical applications. The present work fulfills these needs, focusing on the development of novel epoxide hydrolase as versatile biocatalyst. Epoxide hydrolase enzyme from the newly isolated bacterium Achromobacter sp. MTCC 5605 is much more enantioselective than any other known bacterial epoxide hydrolases